SEASONAL ENERGY RHEOSTASIS: LESSONS FROM THE GROUND SQUIRREL

Abstract

How animals hibernate has been a question in biology since it was first recorded by Aristotle in 350 BCE, upon the observation of animals “concealing” themselves in warm places during the winter. Perhaps two of the most notable, yet equivocal physiological changes associated with hibernation (aside from the act of hibernation itself) are (1) how animals can rapidly gain body mass to withstand an extended dormant period, and (2) how some animals terminate hibernation reproductively competent. Many biologists assume that animals must maintain some physiological stability around biological set-points (i.e., homeostasis) throughout their lifetime. Contrarily, seasonally hibernating animals alter levels of physiological stability, i.e., change homeostatic set-points across space and time (i.e., rheostasis). How animals transition between these seasonal physiological states remains a puzzle.In Chapter 1 of this dissertation, I provide a physiological and conceptual summary of the mechanisms surrounding seasonal adiposity in vertebrates. I start by discussing the adaptive significance of seasonal fluctuations in body mass in vertebrates. I then discuss current models for body weight regulation and whether these may be adapted/modified to help us understand how seasonal vertebrates regulate fluctuations in body mass throughout the year. I discuss the various modes of time measurement in seasonal vertebrates, as some rely on changes in daylength to induce physiological changes, while others undergo self-sustaining endogenous rhythms that are sufficient to drive physiological transitions. Finally, I introduce three different hypotheses regarding the molecular control mechanisms that underlie seasonal energy homeostasis. These hypotheses draw upon evidence from rodent biomedical models and from laboratory and field studies of seasonal vertebrates known to display programmed fluctuations in adiposity across the year. As part of a ground squirrel’s circannual rhythms, every springtime, upon hibernation emergence, animals allocate energy towards reproductive organ development and mate with conspecifics. However, if fat stores are too low upon hibernation termination, animals cannot allocate energy towards reproductive organ development; thus, fall-time gains in fat have fitness consequences. In Chapter 2, I investigate how the hypothalamus changes over the course of hibernation, in preparation for springtime emergence and reproductive competence. In Arctic ground squirrels, males terminate hibernation before females and undergo reproductive development and spermatogenesis over several weeks after body temperature returns to euthermic levels. Conversely, females end hibernation later than males but are reproductively receptive within 48h, such that copulation begins shortly after emergence. Recent research in Arctic ground squirrels has shown drastic remodeling of signaling pathways in hypothalamic regions of the brain. Thus, given these differences in hibernation phenology, I predicted that there may be significant differences between the sexes at the level of the hypothalamic transcriptome. I performed bulk RNA-seq on the hypothalamus at early and late hibernation and found substantial sex differences in gene expression, as well as important changes in transcriptional regulators across hibernation. Many of these transcriptional changes were related to the transport of hormones and neurogenic processes, and these were more apparent in females. Genes that were differentially expressed across hibernation are known to be predominantly expressed in third ventricle glial cells (i.e., tanycytes), supporting a model in which annual changes in gene expression rely on a progressive remodeling of tanycytes across hibernation. For Chapter 3, I focus on the neurobiological and physiological mechanisms responsible for late summer/fall-time fattening (hyperphagia) and the subsequent shift to food suppression/body mass loss (i.e., hypophagia) that occurs immediately before the onset of hibernation. A paradoxical phenomenon in ground squirrels is the observation that animals reach peak body mass, but exhibit a short, 1–2-week period where animals cease feeding, begin to lose body weight, but do not show signs of torpor/hibernation readiness. However, the regulatory mechanisms underlying seasonal fattening in ground squirrels are equivocal. I first quantified daily food consumption and body weight across the fattening period in lab-held 13-lined ground squirrels. Similar to studies in free-living ground squirrels, I found that animals reach a plateau in body mass; however, while animals were fattening, daily food intake decreased. I subsequently measured overnight metabolic rate and internal body temperature and showed that animals gain mass in the late summer/fall by reducing energy expenditure (i.e., seasonal reductions in body temperature and oxygen consumption). Prior studies in seasonal mammals report animals being resistant to signals that promote feeding cessation (i.e., anorectic signals). To test this, I infused animals for 7-days with the fat-derived hormone, leptin, and showed that animals are resistant to its effects, i.e., they do not alter food intake or body weight. Finally, to determine the neuroendocrine mechanisms that underlie the switch from hyperphagia to hypophagia, I microdissected a region of the hypothalamus and performed bulk RNA-seq. I found that canonical pathways in the hypothalamus (i.e., thyroid hormone and retinoic acid signaling) are downregulated in hypo- versus hyperphagic animals. I further show that there is a reduction in the expression of genes involved in cilia assembly and GPCR signaling, suggesting that a reprogramming of this metabolic scaffold is important for generating a hypophagic phenotype. Using single-nuclei RNA-seq data of hypothalamic punches, I show that differentially expressed genes identified via bulk RNA-seq are predominantly expressed in glial cells that line the third ventricle of the hypothalamus (i.e., tanycytes). These findings further support tanycyte cells as critical mediators of seasonal transitions in physiology. Altogether, this dissertation advances our understanding of the physiological and mechanistic underpinnings that govern transitions in seasonal biology.